Difference between revisions of "Part:BBa K3525005"
 
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<partinfo>BBa_K3525005 short</partinfo>
 
<partinfo>BBa_K3525005 short</partinfo>
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<partinfo>BBa_K3525005 SequenceAndFeatures</partinfo>
  
 
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Composite part BBa_K3525005 contains the coding sequences of LacZ.  
 
Composite part BBa_K3525005 contains the coding sequences of LacZ.  
 
[[File:T--Shanghai high school- BBa K3525005 fig 0.png|500px|thumb|center|figure 1]]
 
[[File:T--Shanghai high school- BBa K3525005 fig 0.png|500px|thumb|center|figure 1]]
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===Engineering Success===
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We transformed a pET28a plasmid with lacZ into E. coli BL21 (DE3) and induced lacZ expression by IPTG. After the verification of successful expression of lacZ, we used the HIS tag on lacZ when expressed on pET28a plasmid to purify the lacZ protein by nickel column. We obtained the purified lacZ protein which is about 110KD by elution with 250mm imidazole (Figure 2).
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[[File:T--Shanghai_high_school-_BBa_K3525005_fig_1.png |500px|thumb|center|figure 2]]
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                                          Figure 2: Expression and purification of lacZ protein
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1: Negative control, 2: Lysate of lacZ expressing BL21(DE3), 3: precipitation of lacZ expression BL21(DE3), 4: supernatant of lacZ expression BL21(DE3), 5: Nickel column flow-through fluid of lacZ expression BL21(DE3), 6: 15mM imidazole eluent, 7: 250mM imidazole eluent
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Then we identified the purified lacZ protein by X-gal (Figure 3) and ONPG (Figure 4) respectively. The results showed that both reactions could detect lacZ, and the bright yellow color reaction of ONPG was more obvious from the naked eye. At the same time, we confirmed that the optimum reaction temperature of lacZ with ONPG is 37℃ (Figure 4A). And the reaction can be quantified by detecting the absorbance at 420nm (Figure 4B).
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[[File:T--Shanghai high school- BBa K3525005 fig 2.png|500px|thumb|center|figure 3]]
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                              Figure 3: Identification of purified lacZ protein by adding different amount of x-gal
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[[File:T--Shanghai high school- BBa K3525005 fig 3.png|500px|thumb|center|figure 4A]]
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[[File:T--Shanghai high school- BBa K3525005 fig 4.png |500px|thumb|center|figure 4B]]
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                    Figure 4: Identification of purified lacZ protein by reacting with ONPG at different temperatures.
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A: Color reaction of lacZ with ONPG, B: Absorption values of lacZ and ONPG color reaction at 420nm

Latest revision as of 08:43, 27 October 2020

T7 pro-His-lacZ-His-T7 ter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

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Contribution

Composite part BBa_K3525005 contains the coding sequences of LacZ.

figure 1

Engineering Success

We transformed a pET28a plasmid with lacZ into E. coli BL21 (DE3) and induced lacZ expression by IPTG. After the verification of successful expression of lacZ, we used the HIS tag on lacZ when expressed on pET28a plasmid to purify the lacZ protein by nickel column. We obtained the purified lacZ protein which is about 110KD by elution with 250mm imidazole (Figure 2).

figure 2
                                         Figure 2: Expression and purification of lacZ protein

1: Negative control, 2: Lysate of lacZ expressing BL21(DE3), 3: precipitation of lacZ expression BL21(DE3), 4: supernatant of lacZ expression BL21(DE3), 5: Nickel column flow-through fluid of lacZ expression BL21(DE3), 6: 15mM imidazole eluent, 7: 250mM imidazole eluent Then we identified the purified lacZ protein by X-gal (Figure 3) and ONPG (Figure 4) respectively. The results showed that both reactions could detect lacZ, and the bright yellow color reaction of ONPG was more obvious from the naked eye. At the same time, we confirmed that the optimum reaction temperature of lacZ with ONPG is 37℃ (Figure 4A). And the reaction can be quantified by detecting the absorbance at 420nm (Figure 4B).

figure 3
                              Figure 3: Identification of purified lacZ protein by adding different amount of x-gal
figure 4A
figure 4B
                    Figure 4: Identification of purified lacZ protein by reacting with ONPG at different temperatures.

A: Color reaction of lacZ with ONPG, B: Absorption values of lacZ and ONPG color reaction at 420nm